The project proposed directly targets the vexing problem of controlling aberrant inflammation-induced carcinogenic signals (e.g., TLR4) in the intestine by understanding the biological properties of adopted orphan nuclear receptors (ONRs). One of these receptors, Pregnane X Receptor (PXR), is abundantly expressed in the intestines and is a potential therapeutic target for colitis ?associated colon cancer (CRC). Since existing treatments for colitis-associated CRC are limited and have significant side effects, non-toxic targeting of validated biological targets to prevent colon cancer is warranted. Based on our novel observations that IPA abrogates murine intestinal inflammation (IBD) directly through the non-hematopoietic Pregnane X Receptor (PXR)/Toll-like Receptor 4 (TLR4) signaling pathway, the goal of this project is to test the hypothesis that intestinal PXR can be uniquely modulated by small molecules designed to mimic the gut indole metabolites as a novel approach to treat IBD. Based on the indole/IPA chemical scaffold mimicry, this project will generate novel PXR ligands that can therapeutically target intestinal inflammation and colon cancer in humans, and will provide mechanistic insights into how these molecules binds to PXR. Our preliminary studies have shown that IPA derived from symbionts significantly reduces indomethacin-induced intestinal injury in mice in a PXR and TLR4 dependent manner. IPA regulates intestinal barrier function through PXR. An inverse relationship between PXR and TLR4 as well as IPA and inflammation in human intestinal samples and cell lines, supports our findings in mice. In mice, IPA is a potent activator of PXR, while the human receptor is effectively activated when combined with base indole at a physiologically relevant level. Human PXR LBD mutants were insensitive to activation by indole and IPA. Both Indole and IPA bind to PXR protein in solution. IPA protects against colitis-induced CRC in mice. Small molecule mimics (FKK) of indole/IPA chemical scaffold potently activate PXR and are non-toxic to cells and tissues. Thus, as PXR is a relevant target for intestinal inflammation, we hypothesize that microbial metabolite mimicry will allow for the design of novel, potent and most of all safe compounds that activate PXR and abrogate colitis-associated CRC. To achieve our goals we will (1) synthesize and validate in vitro FKK drug-like lead compounds targeting PXR using rational structure based design; (2) optimize lead FKK candidates based on binding affinity and specificity; (3) evaluate the in vivo efficacy of the lead FKK compounds in abrogating CRC using chemical hPXR mouse models of intestinal inflammation/CRC. In the short-term, we hope to have validated a single novel therapeutic lead based on their likelihood to safely abrogate CRC in mice. These studies can serve as the basis for further validation in human disease-specific animal models in the laboratory prior to embarking on clinical translation. Since PXR has been shown to significantly modulate barrier function in mice, our IPA-like leads could potentially have broader impact on other diseases propelled by a dysfunctional intestinal barrier.